Determination of the Absolute Configurations and the Sensory

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Chemistry and Biology of Aroma and Taste

Determination of the Absolute Configurations and the Sensory Properties of the Enantiomers of a Homologous Series (C6–C10) of 2-Mercapto-4-alkanones Christiane Kiske, Anja Devenie Riegel, Ronja Hopf, Anna Kvindt, Iulia Poplacean, Tohru Taniguchi, Mahadeva M. M. Swamy, Kenji Monde, Wolfgang Eisenreich, and Karl-Heinz Engel J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06599 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 3, 2019

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Journal of Agricultural and Food Chemistry

Determination of the Absolute Configurations and the Sensory Properties of the Enantiomers of a Homologous Series (C6–C10) of 2-Mercapto-4-alkanones

Christiane Kiske†, Anja Devenie Riegel†, Ronja Hopf†, Anna Kvindt†, Iulia Poplacean†, Tohru Taniguchi‡, Mahadeva M. M. Swamy ‡, Kenji Monde‡, Wolfgang Eisenreich§ and Karl-Heinz Engel†*

†

Technische Universität München, Lehrstuhl für Allgemeine

Lebensmitteltechnologie, Maximus-von-Imhof-Forum 2, D-85354 FreisingWeihenstephan, Germany

‡

Frontier Research Center for Advanced Material and Life Science, Faculty of

Advanced Life Science, Hokkaido University, Kita 21 Nishi 11, Sapporo 001-0021, Japan

§

Technische Universität München, Lehrstuhl für Biochemie, Lichtenbergstraße 4, D-

85747 Garching, Germany

CORRESPONDING AUTHOR FOOTNOTE. * Author to whom correspondence should be addressed [Tel.: +49-(0)8161-71-4250. Fax: +49-(0)8161-71-4259. E-mail: [email protected]]

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Abstract

2

The enantiomers of a homologous series (C6-C10) of 2-mercapto-4-alkanones were

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obtained by lipase-catalyzed kinetic resolution of the corresponding racemic 2-

4

acetylthio-4-alkanones. Their configurations were assigned via vibrational circular

5

dichroism and 1H-NMR anisotropy-based methods. Odor thresholds and odor qualities

6

were determined by capillary gas chromatography/olfactometry using chiral stationary

7

phases. There were minima of the odor thresholds for the chain lengths C7/C8. Except

8

for chain length C8, the enantiomers of the other homologs showed similar odor

9

thresholds. The odor qualities ranged from pungent (C5) to mushroom (C9/10) and

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were similar to those known for the corresponding 1-alken-3-ones with one C-atom

11

less. In contrast to their positional isomers (4-mercapto-2-alkanones), the investigated

12

2-mercapto-4-alkanones do not meet the requirements of a “tropical olfactophore”, i.e.

13

compounds possessing a 1,3-oxygen-sulfur functionality and specific arrangements of

14

the substituents.

15 16

Keywords

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2-mercapto-4-alkanones; absolute configuration; VCD; 1H-NMR spectroscopy; odor

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threshold; odor quality


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Introduction

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Sulfur-containing volatiles play prominent roles for the aroma of many foods.1-3 Among

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these important odorants, polyfunctional thiols are of particular interest, owing to their

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pronounced odor qualities and their low odor thresholds.4,5 Recent systematic studies

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on structure-odor correlations in homologous series of mercaptoalkanols confirmed the

24

potencies and the attractive odor qualities of this class of sulfur-containing aroma

25

compounds.6

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β-Mercaptoalkanones and β-mercaptoalkanols are representatives of polyfunctional

27

thiols which have been reported to occur in cooked red bell pepper7 and in aged

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Cheddar cheese.8,9 Wakabayashi et al.10 investigated the stereoisomers of a

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homologues series (chain lengths C5-C10) of 4-mercapto-2-alkanones and the

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corresponding 4-acetylthio-2-alkanones. The enantiomers have been separated via

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capillary gas chromatography (GC) using chiral stationary phases and the absolute

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configurations have been assigned using the 1H-NMR anisotropy method. Odor

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thresholds and odor qualities of the enantiomers have also been determined.11 For the

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homologous series (C5-C10) of 4-mercapto-2-alkanols, GC separations of the

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stereoisomers as well as assignments of the configurations and assessments of odor

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qualities have been achieved.12,13

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Cooked red bell pepper has been reported to contain not only 4-mercapto-2-heptanone

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and 4-mercapto-2-heptanol but also the positional isomers 2-mercapto-4-heptanone

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and 2-mercapto-4-heptanol.7 For 2-mercapto-4-heptanone, the separation of the

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enantiomers and the assignment of the configurations have been described,14 and the

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distribution of the enantiomers in bell peppers has been determined.15

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The objective of this study was to extend the analytical knowledge elaborated for 2-

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mercapto-4-heptanone to a homologous series (C6-C10) of 2-mercapto-4-alkanones. 3 ACS Paragon Plus Environment


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The absolute configurations of the enantiomers should be assigned and their GC-order

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of elution should be determined. By comparison of the odor thresholds and odor

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qualities of the enantiomers with data available for the homologous series of 4-

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mercapto-2-alkanones, further insight into structure-odor relationships for the class of

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β-mercaptoalkanones should be gained.

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MATERIALS AND METHODS

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Chemicals. 4-Hexen-3-one, 2,2-dimethyl-1,3-dioxan-4,6-dione (Meldrum’s acid),

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hexanoyl chloride, heptanoyl chloride, oxalyl chloride, 4-(dimethylamino)pyridine

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(DMAP), N,N’-dicyclohexylcarbodiimide (DCC), (R)-(-)-2-methoxy-2-phenylacetic acid

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((R)-MPA), lipase from Candida antarctica (B lipase, adsorbed on a macroporous

55

acrylic resin, CAL-B), (E)-2-decenal, and deuterochloroform (CDCl3) were purchased

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from Sigma-Aldrich (Steinheim, Germany). Thioacetic acid was obtained from Merck

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Schuchardt OHG (Hohenbrunn, Germany). 2-Octen-4-one, (S)-(+)-2-methoxy-2-(1-

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naphthyl)propionic acid ((S)-MαNP), and (R)-(-)-2-phenylpropionic acid ((R)-HTA)

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were purchased from TCI Europe (Zwijndrecht, Belgium) and silica gel (NormaSil60,

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40-63 µm) from VWR Chemicals (Leuven, Belgium). CDCl3 used for VCD

61

measurements was purchased from Cambridge Isotope Laboratories (Tewksbury,

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MA).

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Syntheses. 2-Nonen-4-one and 2-decen-4-one. The starting materials methyl 3-

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oxooctanoate and methyl 3-oxononanoate were synthesized according to Oikawa et

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al.16, starting with 69.4 mmol Meldrum’s acid in 70 mL dichloromethane (CH2Cl2). After

66

addition of 138.8 mmol (2.0 equiv.) pyridine and 76.3 mmol (1.1 equiv.) hexanoyl

67

chloride (for C9) or heptanoyl chloride (for C10), the reaction mixture was stirred for

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1 h under ice cooling and for another hour at room temperature (RT) under argon. 4 ACS Paragon Plus Environment

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Subsequently, it was washed with diluted (10%) HCl (3x20 mL) and H2O (2x20 mL),

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dried over anhydrous sodium sulfate, and the solvent was removed using a rotary

71

evaporator. The crude product was mixed with 10 mL of methanol and stirred for 2 h

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under reflux, followed by evaporation of the solvent and column chromatography on

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silica gel with a mixture of n-hexane/Et2O (6+1, v/v). The 3-oxoacid esters were directly

74

used for the alkenone synthesis.7,14 The alkenones were purified by distillation under

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vacuum: 2-Nonen-4-one: boiling point (bp): 78-80 °C (11 mbar), yield (y): 56.0%, purity

76

by GC: (p) 84.7%; linear retention indices (LRI) determined by GC: 1485 (Rtx®-WAX),

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1101 (DB-1); 2-decen-4-one: bp: 83-85 °C (5 mbar), y: 18.9%, p: 76.2%, LRI: 1578,

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1203.

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2-Acetylthio-4-alkanones. 6 and 8-10 (Figure 1) were synthesized by Michael-type

80

addition of thioacetic acid (1.1 equiv.) to the respective alkenones.10,11 2-Acetylthio-4-

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hexanone 6: yield (y): 107.1%, purity by GC (p): 92.4%, LRI: 1822 (Rtx®-WAX), 1225

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(DB-1); 2-acetylthio-4-octanone 8: y: 97.1%, p: 96.0%, LRI: 1989, 1411; 2-acetylthio-

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4-nonanone 9: y: 115.1%, p: 88.0%, LRI: 2093, 1511, and 2-acetylthio-4-decanone 10:

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y: 116.6%, p: 89.1%, LRI: 2198, 1613.

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2-Mercapto-4-alkanones. 1 and 3-5 (Figure 1) were obtained by treatment of the 2-

86

acetylthio-4-alkanones with methanol/sulfuric acid.14 Crude products were purified by

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column chromatography on silica gel by elution with a mixture of n-hexane/Et2O (4+1,

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v/v) for 1, (7+1, v/v) for 3, and (8+1, v/v) for 4 and 5; the obtained fractions were

89

checked by TLC. 2-Mercapto-4-hexanone 1: yield (y): 46.7%, purity by GC (p): 92.1%,

90

LRI: 1471 (Rtx®-WAX), 982 (DB-1); 2-mercapto-4-octanone 3: y: 54.2%, p: 95.7%, LRI:

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1649, 1176; 2-mercapto-4-nonanone 4: y: 89.8%, p: 73.0%, LRI: 1755, 1279; 2-

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mercapto-4-decanone 5: y: 41.8%, p: 93.1%, LRI: 1861, 1383.

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Mass spectrometric and NMR data of the synthesized compounds are given in the

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Supporting Information. 5 ACS Paragon Plus Environment


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Lipase-catalyzed kinetic resolutions. The 2-acetylthio-4-alkanones were mixed with

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50 mM potassium phosphate buffer (pH 7.4), CAL-B was added, and the mixture was

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stirred magnetically with a Teflon stir bar at RT. After defined reaction times, the

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mixture was filtered and extracted with Et2O (3x15 mL). The combined organic layers

99

were dried with anhydrous sodium sulfate, filtered, and the solvent was removed under

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reduced pressure. The formed (R)-2-mercapto-4-alkanones were separated from the

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remaining substrates via column chromatography on silica gel by elution with a mixture

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of n-hexane/Et2O (4+1, v/v) for 1, (7+1, v/v) for 3, and (8+1, v/v) for 4 and 5; the

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obtained fractions were checked by TLC (ALUGRAM® SIL G/UV254, Macherey-

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Nagel, Germany), visualization was achieved by spraying with 10% sulfuric acid and

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subsequent heating until dryness. The remaining (S)-acetylthioalkanone substrates

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were transesterified with 10 mL acidified methanol (pH 1-2) under reflux for 24 h. The

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reaction mixture was cooled to RT, diluted with 30 mL of Et2O, and washed with water

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(3x10 mL). After drying with anhydrous sodium sulfate, the solvent was removed under

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reduced pressure to yield the mercaptoalkanone enantiomers. The employed amounts

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of substrate and enzyme, reaction times, conversion rates, enantiomeric excesses,

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purities, and yields are presented in Table 1.

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Formation of diastereoisomers with chiral auxiliaries. (R)-hydratropic acid (HTA)

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thioesters. The thioesters were synthesized as previously described14 using (R)-HTA

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(1.09 mmol), 0.72 mL oxalyl chloride, and 0.36 mmol of the respective 2-mercapto-4-

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alkanone enantiomer (reaction time 72 h). (R)-HTA thioester 11 of (R)-1: 84.3 mg, yield

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(y): 88.9%; 12 of (S)-1: 64.2 mg, y: 66.7%; 13 of (R)-3: 89.1 mg, y: 86.1%; 14 of (S)-3:

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55.5 mg, y: 52.8%; 15 of (R)-4: 101.8 mg, y: 91.7%; 16 of (S)-4: 74.5 mg, y: 66.7%; 17

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of (R)-5: 92.1 mg, y: 80.6%; 18 of (S)-5: 56.8 mg, y: 50.0%. 1H-NMR data are

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presented in Table 2. 6 ACS Paragon Plus Environment

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(S)- and (R)-MPA thioesters. The derivatization was performed according to Porto et

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al.17 using 0.17 mmol (1.0 equiv.) of the (R)-mercaptoalkanone, 0.21 mmol (1.2 equiv.)

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of (R)- or (S)-MPA, 0.21 mmol (1.2 equiv.) of DCC, and DMAP (5 mg) in 1 mL of dry

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CH2Cl2. The reaction mixtures were stirred for 2 h under RT. After work up, purification

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was achieved by semi-preparative HPLC. (R)-MPA thioester 19 of (R)-1: 7.0 mg, yield

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(y): 14.4%; (S)-MPA thioester 20 of (R)-1: 14.5 mg, y: 29.8%; (R)-MPA thioester 21 of

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(R)-3: 7.0 mg, y: 13.2%; (S)-MPA thioester 22 of (R)-3: 14.5 mg, y: 27.1%; (R)-MPA

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thioester 23 of (R)-4: 7.7 mg, y: 13.8%; (S)-MPA thioester 24 of (R)-4: 13.9 mg, y:

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24.8%; (R)-MPA thioester 25 of (R)-5: 20.8 mg, y: 35.6%; (S)-MPA thioester 26 of (R)-

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5: 14.3 mg, y: 24.5%.

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(S)-MαNP thioesters. A solution of DCC (2.0 equiv.) and DMAP (1.0 equiv.) in 1 mL

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dry CH2Cl2 was added to a solution of (S)-MαNP (1.0 equiv.) and the respective

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enantiomerically enriched mercaptoalkanone in 1 mL dry CH2Cl2. The mixture was

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stirred at RT for 20 h, filtered through a syringe filter (0.45 µm), and dried under a N2-

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stream. The residue was separated by semi-preparative HPLC. (S)-MαNP thioester 27

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of (R)-1: 3.6 mg, yield (y): 23.1%; 28 of (S)-1: 4.6 mg, y: 23.3%; 29 of (R)-3: 4.8 mg, y:

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29.5%; 30 of (S)-3: 5.5 mg, y: 27.9%; 31 of (R)-4: 7.7 mg, y: 40.4%; 32 of (S)-4: 8.0 mg,

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y: 43.4%; 33 of (R)-5: 8.4 mg, y: 31.4%; 34 of (S)-5: 7.8 mg, y: 29.6%. 1H-NMR data

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are presented in Table 3.

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High Performance Liquid Chromatography (HPLC). Semi-preparative separations

140

of the diastereoisomers of (R)-HTA, (S)- and (R)-MPA, and (S)-MαNP thioesters of

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mercaptoalkanones were carried out on a Dionex HPLC system (UltiMate 3000 series,

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Dionex, Germering, Germany) equipped with a 3100 wavelength detector set at

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254 nm using a 250 x 8 mm i.d. Nucleosil 50-5 column (CS Chromatography,

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Langerwehe, Germany). Isocratic elution was performed at 30 °C with the following 7 ACS Paragon Plus Environment


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eluents and flow rates: for HTA-thioesters 11-16 and MPA-thioesters 19 and 20:

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Hex/EtOAc (90/10, v/v) with 3 mL/min; for HTA-thioesters 17 and 18: Hex/EtOAc

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(85/15, v/v) with 3 mL/min; for MPA-thioesters 21 and 22: Hex/EtOAc 92/8 with

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3.5 mL/min; for MPA-thioesters 23 and 24: Hex/EtOAc 95/5 with 3.5 mL/min; for MPA-

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thioesters 25 and 26: Hex/EtOAc 96/4 with 4.0 mL/min, for MαNP-thioesters 27 and

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28: Hex/EtOAc 10/1 with 4.0 mL/min; for MαNP-thioesters 29 and 30: Hex/EtOAc 20/1

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with 4.0 mL/min; for MαNP-thioesters 31-34: Hex/EtOAc 25/1 with 4.0 mL/min.

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NMR Spectroscopy. CDCl3 was used as solvent. 1H-NMR and 13C-NMR spectra were

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recorded at 500 MHz and 126 MHz, respectively, with Avance 500 spectrometers

154

(Bruker, Billerica, MA, USA). 1H-detected experiments were done with an inverse

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1H/13C

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13C/31P/29Si/19F/1H

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standard parameter sets of the TOPSPIN 3.0 software package (Bruker).

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spectra were recorded in proton-decoupled mode. The spectra were recorded at 27 °C.

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All signals were assigned by proton-proton and proton-carbon correlation experiments

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(e.g. COSY, HSQC, and HMBC). Data processing was typically done with the

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MestreNova software (Mestrelab Research, Santiago de Compostela, Spain).

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VCD Spectroscopy. VCD and IR spectra were measured on a JASCO FVS-6000

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spectrometer (JASCO International Co. Ltd., Tokyo, Japan) for 3000 and 16 scans,

164

respectively. All spectra were recorded in CDCl3 (c = 0.6 M) using a 50-mm BaF2 cell

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at a resolution of 4 cm-1 at ambient temperature. All VCD spectra were corrected by

166

using those measured for the enantiomers of each sample, and all IR spectra were

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corrected using a solvent spectrum obtained under the identical experimental

168

conditions. The resultant VCD and IR spectra are presented in Δε and ε (both in M-1

169

cm-1) units, respectively.

probehead. Direct

13C-measurements

were performed with a QNP

cryoprobe. The experiments were done in full automation using


13C-NMR

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Computation. Molecular Mechanics Force Field (MMFF) MonteCarlo search was

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performed on SPARTAN’10 software18 and density functional theory (DFT) calculation

172

was carried out on Gaussian 09 package.19 All calculations were conducted without

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considering solvent effects. Theoretical calculations of VCD and IR spectra started with

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a preliminary MMFF search using the arbitrarily selected (R)-enantiomer of 2-

175

mercapto-4-hexanone 1. The obtained conformers within 16 kJ/mol from the most

176

stable were further optimized using the DFT/B3LYP/6-311+G(2df,2p) level of theory.

177

VCD and IR spectra of the resultant 8 stable conformers within a 1.8 kcal/mol window

178

were calculated at the DFT/B3LYP/6-311+G(2df,2p) level and simulated with

179

Lorentzian lineshapes of 6 cm-1 width. The calculated frequencies ν were scaled with

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a factor of 0.98. Final spectra were obtained based on the Boltzmann population

181

average of the spectra of each conformer.

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GC Analyses. Capillary Gas Chromatography (GC-FID). The column used was a 30 m

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x 0.25 mm i.d.; 0.5 µm film thickness (df) Rtx®-Wax (Restek, Bad Homburg, Germany)

184

installed into a HP5890 A gas chromatograph (Hewlett-Packard INC, Waldbronn,

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Germany) equipped with a split/splitless injector (215 °C, split ratio of 1:10) and an FID

186

(300 °C); temperature program: from 40 °C (5 min hold) to 240 °C (30 min hold) at

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4 °C/min; carrier gas: hydrogen at a constant pressure of 150 kPa.

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A 30 m x 0.25 mm i.d.; 1.0 µm df DB-1 column (J&W Scientific, Waldbronn, Germany)

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was installed into a 6890N instrument (Agilent Technologies, Waldbronn, Germany)

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equipped with a split/splitless injector (230 °C, split ratio of 1:10) and an FID (300 °C);

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temperature program: from 60 °C (5 min hold) to 250 °C (5 min hold) at 5 °C/min;

192

carrier gas: hydrogen at a constant pressure of 72 kPa.

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Linear retention indices (LRI) were determined according to van den Dool and Kratz20

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using C8-C40 n-alkane standard solutions (Sigma Aldrich). 9 ACS Paragon Plus Environment


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Enantioselective analysis. For the enantioselective analysis of 2-mercapto-4-

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alkanones and 2-acetylthio-4-alkanones a 25 m x 0.25 mm i.d., 0.25 µm df MEGA-DEX

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DET-Beta column, diethyl tert-butylsilyl-β-cyclodextrin (Mega s.n.c., Legnano, Italy),

198

was used in a 6890N chromatograph (Agilent Technologies) equipped with a

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split/splitless injector (230 °C, split ratio of 1:10) and an FID (300 °C); carrier gas:

200

hydrogen at a constant pressure of 75 kPa.

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Capillary Gas Chromatography/Olfactometry (GC/O). Sensory analyses were

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performed on a Trace GC Ultra (Thermo Fisher Scientific, Dreieich, Germany)

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equipped with a cold-on-column injector (35 °C), a heated sniffing port (200 °C) and

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an FID (250 °C); carrier gas: hydrogen at a constant pressure of 75 kPa. The effluent

205

was split 1:1 via a press-fit Y-splitter and 30 cm x 0.25 mm i.d. deactivated fused silica

206

capillaries (BGB Analytik AG, Rheinfelden, Germany) among sniffing port and FID. For

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compounds 1-10, a 25 m x 0.25 mm i.d., 0.25 µm df MEGA-DEX DET-Beta column,

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diethyl tert-butylsilyl-β-cyclodextrin (Mega s.n.c.), and for 4-mercapto-2-pentanone and

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4-acetylthio-2-pentanone, a 30 m x 0.25 mm i.d.; 0.25 µm df CycloSil-B column, 30 %

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heptakis(2,3-di-O-methyl-6-O-tert-butyl dimethylsilyl)-β-cyclodextrin in DB-1701 (J&W

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Scientific), were installed. The temperature programs used are given in the Supporting

212

Information.

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The sensory analyses of the mercapto- and acetylthio-enantiomers were performed by

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three panelists (females, 22-37 years old). The odor thresholds of the mercapto-

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enantiomers were additionally evaluated by a fourth panelist (panelist 4, female, 21

216

years old). Panelists 1 and 4 had no prior experience with GC/O assessments,

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whereas panelist 2 and 3 were experienced. Odor thresholds in air were determined

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according to the procedure described by Ullrich and Grosch using (E)-2-decenal with

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the reported odor threshold of 2.7 ng/L in air as internal standard.21,22 Known amounts 10 ACS Paragon Plus Environment

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of the internal standard, of 2-mercapto-4-alkanones, and of 2-acetylthio-4-alkanones

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were dissolved in Et2O and diluted stepwise (1+1, (v/v)). The aliquots were analyzed

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by GC/O until no odor was perceivable. The panelists considered a concentration level

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only as odor threshold if it was the lowest dilution step at which the odor was

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consistently perceived in three consecutive GC/O-runs.13 Flavor dilution factors (FD)

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of the internal standard and of the target compounds were obtained by aroma extract

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dilution analysis (AEDA).23 The odor qualities were determined at one dilution step

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above the odor threshold. Threshold values were determined in duplicate analysis by

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panelists 2 (except C5) and 4; mean values were calculated.

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Gas Chromatography-Mass Spectrometry (GC-MS). A 30 m x 0.25 mm i.d.; 0.5 µm

230

df DB-WAXetr fused silica capillary column (J&W Scientific) installed into a

231

GC 8000TOP gas chromatograph (CE Instruments, Hindley Green, United Kingdom)

232

and directly coupled to a Fisons MD8000TOP mass spectrometer (Fisons Instruments,

233

Manchester, UK) was used for compound identifications. The temperature was

234

programmed from 40 °C (5 min hold) to 240 °C (25 min hold) at 4 °C/min. A

235

split/splitless injector (220 °C, split ratio 1:50) was used, the carrier gas was helium at

236

a constant inlet pressure of 75 kPa. The mass spectra in the electron impact mode (EI)

237

were measured at 70 eV in a scan range from m/z 30 to 250. The source temperature

238

was 200 °C and the interface temperature 240 °C. Data acquisition was done via

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Xcalibur software, version 1.4 (Thermo Fisher Scientific).

240

Determination of Optical Rotations. A Polartronic-E polarimeter (Schmidt &

241

Haensch, Berlin, Germany) fitted with a measuring cell (path length 1 dm) and a

242

sodium lamp (wavelength 589 nm) was used. Samples were diluted in ethanol and

243

measurements were performed at 24 °C. (S)-1: [α]D +59.4, concentration (c):

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3.25 g/100 mL, GC purity (p): 98.3%, enantiomeric excess (ee): 84.4%; (R)-1: [α]D 11 ACS Paragon Plus Environment


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55.3, c: 2.18, p: 96.8, ee: 92.9; (S)-3: [α]D +55.9, c: 1.96, p: 97.3, ee: 88.6; (R)-3: [α]D -

246

25.0, c: 0.86, p: 90.7, ee: 92.9; (S)-4: [α]D +50.6, c: 1.49, p: 97.8, ee: 94.2; (R)-4: [α]D -

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47.1, c: 1.46, p: 95.6, ee: 91.7; (S)-5: [α]D +45.0, c: 1.48, p: 96.7, ee: 93.1; (R)-5: [α]D -

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42.1, c: 1.23, p: 84.3, ee: 94.6; (S)-6: [α]D -11.6, c: 2.22, p: 97.0, ee: 99.6; (S)-8: [α]D -

249

16.5, c: 1.52, p: 96.7, ee: 99.6; (S)-9: [α]D -18.4, c: 1.07, p: 91.2, ee: 96.4; (S)-10: [α]D

250

-18.4, c: 1.44, p: 95.0, ee: 97.9;

251 252

RESULTS AND DISCUSSION.

253

In analogy to the procedure previously described for 2-mercapto-4-heptanone 2, 2-

254

mercapto-4-alkanones with carbon chain lengths C6 to C10 (1 and 3-5, Figure 1) were

255

synthesized by Michael-type addition of thioacetic acid to the respective alkenones and

256

subsequent treatment of the formed 2-acetylthio-4-alkanones (6 and 8-10, Figure 1)

257

with methanol/sulfuric acid.7,14 GC separations of the enantiomers of the homologous

258

series of 2-mercapto-4-alkanones (Figure 2A) and 2-acetylthio-4-alkanones (Figure

259

2B) were achieved using diethyl tert-butylsilyl-β-cyclodextrin as chiral stationary phase.

260

Determination of the Absolute Configurations. Enantiomerically enriched 2-mercapto-

261

4-alkanones were obtained by kinetic resolutions of the respective racemic 2-

262

acetylthio-4-alkanones using the lipase CAL-B as biocatalyst. The employed

263

procedure is exemplarily shown for 2-mercapto-4-octanone 3 in Figure 3. Yields and

264

optical purities of the enantiomers of 2-mercapto-4-alkanones obtained by this

265

approach are summarized in Table 1.

266

The absolute configurations and the orders of elution of the enantiomers of 2-

267

mercapto-4-alkanones and 2-acetylthio-4-alkanones with chain lengths C6 and C8-

268

C10 were determined based on the procedures previously applied to 2-mercapto-4-


Page 12 of 41

Page 13 of 41


269

heptanone, i.e. VCD spectroscopy as well as 1H-NMR analyses of diastereoisomeric

270

thioesters using different chiral auxiliaries.14

271

As a first step, the obtained enantiomers were subjected to VCD experiments. A

272

preliminary

273

311+G(2df,2p) optimization were conducted for the arbitrarily chosen (R)-enantiomer

274

of 2-mercapto-4-hexanone 1, resulting in 8 stable conformers within 1.8 kcal/mol of the

275

most stable one. VCD and IR spectra were calculated for these conformers at the

276

DFT/B3LYP/6-311+G(2df,2p) level of theory. The final VCD and IR spectra were

277

obtained based on the Boltzmann population average of the spectrum of each

278

conformer (Figure 4A and B). Experimental VCD and IR spectra of 1-E1 were obtained

279

as CDCl3 solutions, at concentrations of 0.60 M. The peak positions and the signs of

280

each signal of the observed VCD spectrum showed excellent agreement with those

281

calculated for (R)-1. Therefore, this VCD result concludes the absolute configuration

282

of 1-E1 as (R).

283

Assuming that the elongation of the carbon chain length from C6 to C10 did not affect

284

their conformations, the VCD and IR spectra of 3-E1, 4-E1 and 5-E1 (also measured

285

in CDCl3 solutions, at the same concentration as 1) were superimposed on those

286

calculated for 1-E1. The observed VCD similarities strongly suggested the absolute

287

configurations of 3-E1, 4-E1, and 5-E1 as (R).

288

Using the method described by Helmchen and Schmierer24, 2-mercapto-4-alkanone

289

enantiomers obtained via lipase-catalyzed kinetic resolution were reacted with (R)-

290

hydratropic acid chloride. As examples, 1H-NMR spectra of (R)-HTA thioesters 11 and

291

12 of (R)-1 and (S)-1, respectively, are shown in Figures 5 A and B. The protons of the

292

methyl group (C-6) as well as both methylene bridges (C-3 and C-5) of (R)-HTA

293

thioester 11 showed a relative upfield shift compared to the protons of (R)-HTA

294

thioester 12. In contrast, the protons of the other terminal methyl group (C-1) of (R)-

MMFF

conformational

search

and


subsequent

DFT/B3LYP/6-


295

HTA thioester 11 underwent a downfield shift compared to the equivalent protons of

296

(R)-HTA thioester 12. The described shifting effects can be attributed to a cisoid

297

arrangement of the acid’s phenyl ring in the depicted constellations of (R)-HTA

298

thioester 11 and 12. Therefore, the (R)-HTA thioester 11 was confirmed as (R,R)

299

whereas the (R)-HTA thioester 12 was confirmed as (R,S). In Table 2, 1H-NMR data

300

of (R)-HTA thioesters 11-18 of the enantiomers of 1 and 3-5 are shown.

301

As second chiral derivatizing reagent, 2-methoxy-2-phenylacetic acid (MPA) was

302

applied. Porto et al.17,25 have previously shown its suitability for the assignment of the

303

absolute configurations of chiral thiols. The ΔδRS signs determined for the (R)- and (S)-

304

MPA thioesters obtained after derivatization of the (R)-enantiomers of 1 and 3-5 were

305

consistent within the homologous series as shown in Figure 6. According to the model

306

developed by Porto et al.25, the spatial arrangement of the L1/L2 side chains confirmed

307

the (R)-configuration of the 2-mercapto-4-alkanones.

308

Finally, (S)-MαNP was used as chiral auxiliary. The (R)- and (S)-enantiomers obtained

309

after lipase-catalyzed kinetic resolution were reacted with (S)-MαNP, purified by semi-

310

preparative HPLC, and analyzed by 1H-NMR spectroscopy (Table 3). Applying the

311

revised sector rule for β-mercaptoalkanones and thiols14 to the (S)-MαNP thioester of

312

1, the Δδ value of H-1 is negative (-0.01) and is placed on the right side whereas the

313

Δδ values for H-3, H-5, and H-6 are positive (0.04, 0.04, and 0.03, respectively) and

314

are placed on the left side. This resulted in (R)-configuration at the C-2 position of the

315

first eluting compound (corresponding to LC-peak I, 27, Table 3). The same results

316

were found for the (S)-MαNP thioesters of 3-5.

317

Based on the consistent results obtained by VCD and NMR analyses, the GC-order of

318

elution of the enantiomers of 2-mercapto-4-alkanones (1 and 3-5) as well as of 2-

319

acetylthio-4-alkanones (6 and 8-10) on the employed chiral stationary phase could be

320

assigned as (S) before (R), (Figure 2A and B). 14 ACS Paragon Plus Environment

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321

Determination of Odor Thresholds. Odor thresholds of the enantiomers of 2-mercapto-

322

4-alkanones (1-5) and 2-acetylthio-4-alkanones (6-10) were determined via GC/O

323

using the method described by Ullrich and Grosch21. Table 4 shows the results

324

obtained for the two homologous series from C6 to C10 as well as for the C5-homologs

325

4-mercapto-2-pentanone and 4-acetylthio-2-pentanone. The mercaptoalkanones were

326

assessed by four panelists, the acetylthioalkanones by three. Regarding their

327

variability, panelist 4 showed a high sensitivity for the C5 and C7 homologs, e.g. factor

328

49 for (S)-4-mercapto-2-pentanone compared to panelist 3 or factor 13 for (S)- and

329

(R)-2-mercapto-4-heptanone 2 compared to panelist 1. However, for the other

330

compounds and panelists the individual odor thresholds either were the same or

331

differed only by factors between 2 and approximately 4. This corresponds to one or

332

two dilution steps in the course of the AEDA and thus indicates the reproducibility of

333

the sensory assessments.

334

A graphic presentation of the geometric means of the odor thresholds depending on

335

the chain lengths is given in Figure 7 (solid lines). The data illustrate that there were

336

minima of the odor thresholds for the chain lengths C7/C8 for both 2-mercapto-4-

337

alkanones (Figure 7A) and 2-acetylthio-4-alkanones (Figure 7B). The graphs for the

338

enantiomers were quite similar; significantly lower odor thresholds were only observed

339

for the (R)-enantiomers of the C8-homologs. This is confirmed by a comparison of the

340

geometric means for the enantiomers shown in Table 4. Except for 2-mercapto-4-

341

octanone (factor 3) and 2-acetylthio-4-octanone (factor 7), the geometric means of the

342

odor thresholds calculated for the enantiomers were either the same or differed only

343

by factors up to 2 (Table 4).

344

Figures 7A and B (dotted lines) also show the odor thresholds previously reported for

345

the homologous series of 4-mercapto-2-alkanones and 4-acetylthio-2-alkanones.11

346

The range of odor thresholds (0.1-2 ng/L) previously described for 4-mercapto-215 ACS Paragon Plus Environment


347

pentanone is in the same order of magnitude as those determined in this study (0.1-

348

4.9 ng/L). The C8 and C9 homologs of 4-mercapto-2-alkanones showed lower odor

349

threshold values than the respective 2-mercapto-4-alkanones; another striking

350

difference was the high odor threshold determined for (S)-4-mercapto-2-hexanone.

351

The odor thresholds of 4-acetylthio-2-pentanone (7-44 ng/L) determined by the

352

panelists in this study were significantly lower than those previously reported (70-200

353

ng/L).11 The odor thresholds of (R)-2-acetylthio-4-octanone and the 2-acetylthio-4-

354

decanone enantiomers were significantly lower than those of the respective 4-

355

acetylthio-2-alkanone positional isomers. Except for these differences, the shapes of

356

the curves depending on the chain lengths showed similar minima and maxima and

357

were comparable for the positional isomers. The significant differences in odor

358

thresholds between the mercaptoalkanones and the respective acetylthioalkanones

359

were similarly expressed for the homologous series of both positional isomers.

360

Determination of Odor Qualities. Odor qualities of the enantiomers of 2-mercapto-4-

361

alkanones and 2-acetylthio-4-alkanones were determined via GC/O at one dilution step

362

above the odor threshold (Tables 5 and 6). It is striking that the fruity and sulfury-catty

363

notes reported for the (R)- and (S)-enantiomers of 4-mercapto-2-alkanones,

364

respectively, were not observed for 2-mercapto-4-alkanones. As summarized in Table

365

5, their odor qualities changed, depending on the chain length, from pungent, plastic

366

(C6/C7) and roasty (C8) to earthy and mushroom (C9 and C10). No consistent

367

differences between the (R)- and (S)-enantiomers were observed.

368

Structural requirements for a “tropical olfactophore” have been proposed by Rowe26

369

and were extended to the respective acetyl compounds by Robert et al.27 (Figure 8A).

370

In contrast to this model, the 2-mercapto-4-alkanones and 2-acetylthio-4-alkanones

371

(chain lengths C6–C10) possess alkyl groups (from ethyl to hexyl) rather than 16 ACS Paragon Plus Environment

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372

hydrogen, a methyl group, a ring system, or an ether moiety as the substituent R4. This

373

may explain the lack of tropical, fruity notes. Among the β-mercaptoalkanones

374

investigated in this study, only the C5 homolog 4-mercapto-2-pentanone meets the

375

structural requirements suggested by Rowe;26 this is in line with the descriptions “fruity”

376

by two of the panelists (Table 5).

377

In contrast, within both homologous series the C9 and C10 homologs exhibited

378

pronounced earthy and mushroom odor notes. Figure 8B shows the structural similarity

379

between 2-mercapto-4-nonanone 4 and the known mushroom odorant 1-octen-3-one.

380

The lengths of the alkyl chains (corresponding to the substituent R4 in Figure 8A) are

381

identical; the β-mercapto group in 2-mercapto-4-nonanone 4 seems to play the role of

382

the terminal double bond in 1-octen-3-one. This is strongly supported by the fact that

383

the odor qualities determined for the other 2-mercapto-4-alkanones 1-3 and 5 are also

384

in very good agreement with odor qualities described for the respective homologous

385

series of 1-alken-3-ones.28 The odor thresholds of the homologous 1-alken-3-ones28

386

are significantly lower than those determined for the 2-mercapto-4-alkanones in this

387

study. However, the odor descriptions reported for 1-alken-3-ones in the literature, e.g.

388

pungent and plastic for 1-penten-3-one and 1-hexen-3-one28-31, vegetable-like for 1-

389

hepten-3-one28, and mushroom for 1-nonen-3-one28-34, nearly perfectly match those

390

determined in this study for the corresponding 2-mercapto-4-alkanones with one C-

391

atom more. Preliminary sensory assessments of a homologous series of 2-mercapto-

392

4-alkanols indicated that this phenomenon might also apply to the corresponding

393

alcohols (data not shown). This observation is in agreement with the role of (R)-1-

394

octen-3-ol as another key mushroom aroma compound35,36 and is supported by the

395

description of mushroom-like odors also for 1-mercapto-3-octanol and 1-mercapto-3-

396

nonanol.6



397

The 2-acetylthio-4-alkanones showed mainly vegetable notes for the (R)-enantiomers

398

(C6-C8). Within the homologous series, the predominating notes also changed to

399

earthy and mushroom for 2-acetylthio-4-nonanone and 2-acetylthio-4-decanone,

400

indicating that the phenomenon is also valid for the acetyl-compounds.

401

The flavor of 2-mercapto-4-heptanone 2 tasted in NaCl and sugar solutions has been

402

described as grapefruit, sesame, earthy, and rocket.7 The main descriptions

403

associated with this homolog in the course of the GC/O evaluation were vegetables,

404

onion, and pungent. However, it is noteworthy that one of the panelists mentioned bell

405

pepper as a descriptor for both enantiomers. In cooked bell pepper, (S)-2-mercapto-4-

406

heptanone has been determined as the predominating enantiomer.15 A determination

407

of the odor threshold in water and a calculation of the odor activity value would be

408

required to assess its actual contribution to the aroma of bell peppers.

409

In conclusion, the sensory assessments showed that in contrast to the data reported

410

for the homologous series of 3-mercapto-2-methylalkanols and 1-mercapto-2-methyl-

411

3-alkanols6, the exchange of the positions of the functional groups (SH and carbonyl)

412

in β-mercaptoalkanones did not cause significant changes in the GC-odor thresholds.

413

However, there is a significant impact of the positions of the functional groups on the

414

odor qualities.

415 416

Acknowledgement

417

We thank Christine Schwarz for recording the NMR spectra.

418 419

Supporting Information

420

MS and NMR data of 2-acetylthio-4-alkanones and 2-mercapto-4-alkanones (C6-C10)

421

as well as 2-nonen-4-one and 2-decen-4-one. 1H-NMR data and Δδ values of (R)- and 18 ACS Paragon Plus Environment

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422

(S)-MPA thioesters of (R)-(-)-2-mercapto-4-hexanone 1. 1H-NMR data and Δδ values

423

of (R)- and (S)-MPA thioesters of (R)-(-)-2-mercapto-4-octanone 3. 1H-NMR data and

424

Δδ values of (R)- and (S)-MPA thioesters of (R)-(-)-2-mercapto-4-nonanone 4. 1H-NMR

425

data and Δδ values of (R)- and (S)-MPA thioesters of (R)-(-)-2-mercapto-4-decanone

426

5. 13C-NMR data of synthesized diastereoisomeric derivatives. Temperature programs

427

used for the separations of 2-acetylthio-4-alkanones and 2-mercapto-4-alkanones (C6-

428

C10) as well as 4-acetylthio-2-pentanone and 4-mercapto-2-pentanone.

429

This material is available free of charge via the Internet at http://pubs.acs.org.



430

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540

(35)

541

various species of edible mushrooms. Food Chem. 2004, 86, 113-118.

542

(36)

543

of 1-octen-3-ol enantiomers. J. Agric. Food Chem. 1986, 34, 119-22.

Roberts, D. D.; Acree, T. E. Effects of Heating and Cream Addition on Fresh

Schnermann, P.; Schieberle, P. Evaluation of Key Odorants in Milk Chocolate

Cullere, L.; Fernandez de Simon, B.; Cadahia, E.; Ferreira, V.; Hernandez-Orte,

Zawirska-Wojtasiak, R. Optical purity of (R)-(-)-1-octen-3-ol in the aroma of

Mosandl, A.; Heusinger, G.; Gessner, M. Analytical and sensory differentiation


Page 24 of 41

Page 25 of 41


Figure Captions 545

Figure 1.

546

Structures of investigated 2-mercapto-4-alkanones 1-5 and the respective 2-acetylthio-

547

4-alkanones 6-10.

548

Figure 2.

549

Capillary gas chromatographic separation of the enantiomers of (A) 2-mercapto-4-

550

alkanones 1-5 and (B) 2-acetylthio-4-alkanones 6-10.

551

Figure 3.

552

Preparation of the 2-mercapto-4-octanone enantiomers 3-E1 and 3-E2 via kinetic

553

resolution of 2-acetylthio-4-octanone 8, catalyzed by CAL-B.

554

Figure 4.

555

Comparison of (A) IR and (B) VCD spectra calculated for (R)-2-mercapto-4-hexanone

556

(R)-1 and observed for 1-E1, 3-E1, 4-E1, and 5-E1.

557

Figure 5.

558

1H-NMR

559

12 of (S)-1.

560

Figure 6.

561

Structures of (R)- and (S)-MPA thioesters of (R)-1, (R)-3, (R)-4, and (R)-5 with δ values

562

(ppm) and ΔδRS values (ppm). L1 (front side) and L2 (rear side) correspond to the side

563

chains at the asymmetric centers of the mercaptoalkanone moieties.

564

Figure 7.

565

Geometric means of the odor thresholds of (A) 2-mercapto-4-alkanones: (R)-

566

enantiomer (●), (S)-enantiomer (●) and 4-mercapto-2-alkanones11: (R)-enantiomer

spectra of (R)-hydratropic acid thioesters (A) (R,R)-11 of (R)-1 and (B) (R,S)-



567

(Δ), (S)-enantiomer (Δ) and (B) 2-acetylthio-4-alkanones: (R)-enantiomer (●), (S)-

568

enantiomer (●) 4-acetylthio-2-alkanones11: (R)-enantiomer (Δ), (S)-enantiomer (Δ).

569

Figure 8.

570

(A) Structural requirements for a ‘tropical olfactophore’ as proposed by Rowe26 and

571

extended by Robert et al.27 (A: H, SCH3, ring; B: H, CH3, acyl, absent if carbonyl; R1/R2:

572

H, alkyl; R3: H, alkyl, ring; R4: H, CH3, ring, OR; R5: H, absent if carbonyl. (B) Structures

573

of 2-mercapto-4-nonanone 4 and 1-octen-3-one.


Page 26 of 41

Page 27 of 41


Table 1. Preparation of 2-Mercapto-4-alkanone Enantiomers via Lipase-catalyzed Hydrolysis of the Respective 2-Acetylthio-4alkanones starting compound

obtained enantiomer

configurationb, optical rotation

amountc [g]

lipase CAL-B [g]

volume of buffer [mL]

reaction time [h]

conversion rated [%]

eee [%]

purityd [%]

yieldf [%]

1-E1

(R)-(-)

0.866

1.00

50

0.5

33

92.9

96.8

11.0

1-E2

(S)-(+)

0.689

0.8

40

2

50

84.4

98.3

12.6

3-E1

(R)-(-)

1.947

2.21

110

0.5

33

92.9

98

11.9

3-E2

(S)-(+)

1.36

1.61

95

2

46

88.6

97.3

5.3

4-E1

(R)-(-)

1.029

1.21

60

1

62

91.7

95.6

23.3

4-E2

(S)-(+)

2.130

2.51

150

4

46

94.2

97.8

12.4

5-E1

(R)-(-)

2.132

2.52

150

1

42

94.6

97.1

22.4

5-E2

(S)-(+)

1.068

1.31

70

6

48

93.1

96.7

6.4

a

6

8

9

10

Numbering refers to the enantiomers obtained either as direct hydrolysis product (E1) or via the remaining substrate (E2) Configurations determined via VCD and 1H-NMR analysis of HTA-, MPA-, and (S)-MαNP-thioesters; for optical rotations see Materials and Methods c Used amount of the starting compound d Determined via GC (DBWAX) e Enantiomeric excess, determined via GC (MEGA-DEX DET-Beta) f Molar yields

a b



Page 28 of 41

Table 2. 1H-NMR Data of (R)-HTA Thioesters of the Enantiomers of 2-mercapto-4-hexanone 1, 2-mercapto-4-octanone 3, 2-mercapto4-nonanone 4, and 2-mercapto-4-decanone 5 2-mercapto-4-hexanone 1

2-mercapto-4-octanone 3

2-mercapto-4-nonanone 4

2-mercapto-4-decanone 5

4-E1

4-E2

5-E1

5-E2

(R,R)-15

(R,S)-16

Δδ

(R,R)-17

(R,S)-18

(R)-HTA thioester 1-E1

1-E2

3-E1

3-E2

H

(R,R)-11

(R,S)-12

Δδ

(R,R)-13

(R,S)-14

Δδ

1

1.22 (d, 7.0)

1.18 (d, 7.0)

-0.04

1.22 (s)

1.18 (s)

-0.04

1.22 (d, 7.0)

1.18 (d, 7.0)

-0.04

1.22 (d, 6.9)

1.18 (d,7.0)

-0.04

2

3.81 (m)

3.80 (m)

-0.01

3.80 (m)

3.80 (m)

0

3.80 (m)

3.80 (m)

0

3.81 (m)

3.80 (m)

-0.01

3

2.62 (dd, 16.7, 5.0)

2.68 (dd, 16.7, 5.4)

0.06

2.62 (dd, 16.7, 5.0)

2.68 (dd, 16.7, 5.3)

0.06

2.62 (dd, 16.7, 4.9)

2.68 (dd, 16.7, 5.3)

0.06

2.62 2.68 (dd, 16.7, 4.9) (dd, 16.7, 5.4)

0.06

3'

2.47 (dd, 16.6, 8.3)

2.52 (dd, 16.7, 8.1)

0.05

2.46 (dd, 16.7, 3.3)

2.51 (dd, 16.7, 8.0)

0.05

2.46 (dd, 16.8, 8.3)

2.51 (dd, 16.7, 8.1)

0.05

2.46 2.51 (dd, 16.8, 8.3) (dd, 16.7, 8.1)

0.05

5

2.26 (m)

2.32 (q, 7.4)

0.08

2.24 (td, 7.3, 3.6)

2.29 (dd, 7.9, 7.0)

0.05

2.23 (td, 7.3, 3.7)

2.29 (m)

0.06

2.24 (td, 7.3, 3.7)

2.29 (m)

0.05

6

0.91 (t, 7.3)

0.96 (t, 7.3)

0.05

1.41 (m)

1.46 (m)

0.05

1.43 (m)

1.46 (m)

0.03

1.42 (m)

1.47 (m)

0.05

7

1.18 (t, 7.3)

1.22 (m)

0.04

1.14 (m)

1.15 (m)

0.01

1.17 (m)

1.20 (m)

0.03

8

0.80 (t, 7.3)

0.82 (t, 7.3)

0.02

1.19 (m)

1.22 (m)

0.03

1.17 (m)

1.20 (m)

0.03

0.80 (t, 7.2)

0.81 (t, 7.1)

0.01

1.17 (m)

1.20 (m)

0.03

0.80 (t, 7.1)

0.81 (t, 7.0)

0.01

9 10


Δδ

Page 29 of 41


Table 3. 1H-NMR Data of (S)-MαNP Thioesters of the Enantiomers of 2-mercapto-4-hexanone 1, 2-mercapto-4-octanone 3, 2mercapto-4-nonanone 4, and 2-mercapto-4-decanone 5 2-mercapto-4-hexanone 1

2-mercapto-4-octanone 3

2-mercapto-4-nonanone 4

2-mercapto-4-decanone 5

(S)-MαNP thioester LC Peak Ia

LC Peak IIa

LC Peak Ia

LC Peak IIa

LC Peak Ib

LC Peak IIb

LC Peak Ib

LC Peak IIb

H

(S,R)-27

(S,S)-28

Δδ

(S,R)-29

(S,S)-30

Δδ

(S,R)-31

(S,S)-32

Δδ

(S,R)-33

(S,S)-34

Δδ

1

1.24 (d, 7.0)

1.23 (d, 7.9)

-0.01

1.23 (d, 7.0)

1.22 (d, 7.0)

-0.01

1.23 (d, 6.9)

1.22 (d, 7.0)

-0.01

1.23 (d, 7.0)

1.22 (d, 6.9)

-0.01

2

3.80 (m)

3.81 (m)

-0.01

3.80 (m)

3.79 (m)

-0.01

3.79 (m)

3.80 (m)

0.1

3.79 (m)

3.80 (m)

0.01

3

2.64 (dd, 16.6, 5.0)

2.68 (dd, 16.5, 5.6)

0.04

2.64 (dd, 18.5, 5.1)

2.68 (dd, 16.6, 5.5)

0.04

2.64 (dd, 16.6, 4.8)

2.68 (dd, 16.6, 5.2)

0.04

2.64 (dd, 16.6, 4.9)

2.68 (dd, 16.6, 5.5)

0.04

3'

2.51 (dd, 16.5, 8.5)

2.50 -0.01 (dd, 15.6, 8.1)

2.49 (dd, 16.6, 8.5)

2.49 (dd, 16.5, 8.2)

0

2.49 (dd, 16.6, 8.6)

2.49 (dd, 16.7, 8.2)

0

2.49 (dd, 16.6, 8.6)

2.49 (dd, 16.6, 8.2)

0

5

2.27 (m)

2.31 (m)

0.04

2.24 (td, 7.4, 4.3) 1.41 (m)

2.29 (td, 7.2, 1.8) 1.44 (m)

0.05

2.23 (m)

2.28 (m)

0.05

2.23 (td, 7.5, 4.2)

2.29 (m)

0.06

6

0.91 (t, 7.3)

0.94 (t, 7.3)

0.03

0.03

1.42 (m)

1.46 (m)

0.04

1.41 (m)

1.45 (m)

0.04

7

1.18 (m)

1.19 (m)

0.01

1.16 (m)

1.17 (m)

0.01

1.15 (m)

1.18 (m)

0.03

8

0.78 (t, 7.3)

0.81 (t, 7.4)

0.03

1.16 (m)

1.17 (m)

0.01

1.15 (m)

1.18 (m)

0.03

0.77 (t, 7.2)

0.80 (t, 7.1)

0.03

1.15 (m)

1.18 (m)

0.03

0.78 (t, 6.9)

0.80 (t, 6.8)

0.02

9 10 a Eluent: b Eluent:

hexane/ethyl acetate 20/1 (v/v) hexane/ethyl acetate 25/1 (v/v)



Page 30 of 41

Table 4. Odor Thresholds of the Enantiomers of 2-Mercapto-4-alkanones and 2-Acetylthio-4-alkanones Determined by GC/O odor thresholds in air [ng/L] panelist 1

panelist 2a

panelist 3

panelist 4b

geometric mean ± SDc

no.

compound

(R)

(S)

(R)

(S)

(R)

(S)

(R)

(S)

(R)

(S)

(C5) 1 (C6) 2 (C7) 3 (C8) 4 (C9) 5 (C10)

4-mercapto-2-pentanone 2-mercapto-4-hexanone 2-mercapto-4-heptanone 2-mercapto-4-octanone 2-mercapto-4-nonanone 2-mercapto-4-decanone

4.4 2.5 1.3 0.7 21 39

2.2 0.6 1.3 5.3 10 19

1.1 1.0 0.3 0.2 3.3 16

1.1 2.7 0.4 0.5 5.5 86

4.9 2.4 0.3 0.3 5.2 10

4.9 2.4 2.3 0.6 5.2 10

0.3 0.5 0.1 0.4 3.9 14

0.1 0.6 0.1 0.9 4.9 9.0

1.6 ± 3.8 1.3 ± 2.1 0.3 ± 2.8 0.4 ± 1.6 6.1 ± 2.3 17.2 ± 1.8

1.1 ± 4.6 1.2 ± 2.3 0.6 ± 3.7 1.1 ± 2.9 6.1 ± 1.4 19.6 ± 2.8

(C5) 6 (C6) 7 (C7) 8 (C8) 9 (C9) 10 (C10)

4-acetylthio-2-pentanone 2-acetylthio-4-hexanone 2-acetylthio-4-heptanone 2-acetylthio-4-octanone 2-acetylthio-4-nonanone 2-acetylthio-4-decanone

19 43 37 5.0 149 73

7.0 43 37 21 149 36

15 709 9 11 596 143

39 712 18 86 1192 286

17 22 9.2 2.7 298 71

44 178 18 22 149 71

17 ± 1.1 88 ± 6.3 14 ± 2.3 5 ± 2.0 298 ± 2.0 90 ± 1.5

23 ± 2.8 176 ± 4.1 23 ± 1.5 34 ± 2.2 298 ± 3.3 90 ± 2.9

Mean values calculated from duplicate analysis of mercaptoalkanones with chain lengths C6-C10 Mean values calculated from duplicate analysis of mercaptoalkanones with chain lengths C5-C10 c Geometric standard deviation a b


Page 31 of 41


Table 5. Odor Descriptions of the Enantiomers of 2-Mercapto-4-alkanones Determined by GC/O odor descriptionsa (R)-enantiomer panelist 2

panelist 3

panelist 4

panelist 1

urine, sweat

potatoes, earthy

tallow, cheese, sulfury

vegetables, onion

potatoes, leek, broth

1 (C6)

urine, musk, sweet

urine, sweat

pungent, sulfury

pungent, garlic, onion, savory

2 (C7)

sweet, rotten

vegetables, cabbage

vegetables, pungent, plastic

3 (C8)

urine, sweet, sweat

urine, cooked onion

4 (C9)

mushroom, urine, sweet

5 (C10)

earthy, mushroom, musty, urine

no. (C5)

a

panelist 1

(S)-enantiomer panelist 2

panelist 3

panelist 4

fruity, leek, sweat

fruity, raspberry

sweet, onion, vegetables

citrus

sweat

sweat, dull

sweat, mustard, pungent

vegetables, pungent, onion, bell pepper

sweat, sweet

pungent, onion

vegetables, plastic

onion, vegetables, sweat, pungent, bell pepper

roasty, earthy

cheese, sweet, vegetable broth,

sweet, rotten

roasty, meat

roasty, earthy

vegetable broth, onion, sweat

vegetables, earthy, celery

mushroom, earthy

mushroom, onion, musty

vegetable broth, herbs, sweet

vegetables, mushroom, leek

vegetables, plastic

mushroom, musty, vegetables, earthy

vegetables, dull, onion

mushroom, earthy, musty, dull

mushroom, vegetables, musty

mushroom, dull, herbs, sweet

vegetables, dull

mushroom, vegetables, celery

mushroom, vegetables, earthy, musty,

Determined at one dilution step above the odor threshold; descriptions given by at least two panelists are printed in bold



Page 32 of 41

Table 6. Odor Descriptions of the Enantiomers of 2-Acetylthio-4-alkanones Determined by GC/O odor descriptionsa no.

panelist 1

(C5)

sweat, urine, burnt

6 (C6)

(R)-enantiomer panelist 2

(S)-enantiomer panelist 2

panelist 3

panelist 1

sweat, urine

sweat, tallow

potatoes, leek, sweat

leek, cabbage, dull

tallow, sulfury

vegetables, sweat, sweet

vegetables, sulfury

fennel, dull

sweat, rotten

plastic, burnt

earthy, dull, mushroom

7 (C7)

herbs, savory, sweat, earthy

vegetables, leek

sulfury

sweat, soursweet, herbs

roasty

earthy, sulfury

8 (C8)

earthy, mushroom

vegetables, onion, pungent

burnt, rubber

rubber, rotten

meat, dull

rubber

9 (C9)

earthy, urine, sweet

earthy, cabbage, vegetables,

mushroom

sweat, sweet, herbs

earthy

earthy, rotten mushroom,

earthy, sweat, slightly pungent

mushroom, rubber

mushroom, rancid

mushroom, acidic, pungent

mushroom, earthy

mushroom, greasy/fatty

10 (C10) a

panelist 3

Determined at one dilution step above the odor threshold; descriptions given by at least two panelists are printed in bold


Page 33 of 41


Figures

SH

O R 1-5

O

1 R= methyl 2 R= ethyl 3 R= n-propyl 4 R= n-butyl 5 R= n-pentyl

S

O R 6-10

Figure 1


6 R= methyl 7 R= ethyl 8 R= n-propyl 9 R= n-butyl 10 R= n-pentyl


Figure 2


Page 34 of 41

Page 35 of 41


Figure 3



Figure 4


Page 36 of 41

Page 37 of 41


Figure 5



Figure 6


Page 38 of 41

Page 39 of 41


Figure 7



Figure 8


Page 40 of 41

Page 41 of 41


TOC Graphic


Determination of the Absolute Configurations and the Sensory

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